The present invention relates to apparatus and methods for determining electrolyte concentrations in a patient sample. More particularly, it relates to a new and improved miniaturized reference electrode for use in combination with a miniature ion selective electrode in a multi-chambered sample cartridge for making electrolyte determinations using centrifugal clinical analyzer equipment.
This application is related to the following commonly assigned applications being filed concurrently herewith: Ser. No. 337,007, relating to a miniature ION SELECTIVE ELECTRODE AND METHOD OF MAKING SAME, to be used with the miniature reference electrode of this invention; and two continuations in part from U.S. application Ser. No. 196,120, filed May 25, 1988, the first, Ser. No. 337,011 describing a TWO PART TEST CARTRIDGE FOR CENTRIFUGE, and the second Ser. No. 363,943 relating to digital electronic aspects of an APPARATUS FOR MEASURING ELECTROLYTES. Each of these above-identified applications are specifically incorporated herein by reference.
Methods for determining the electrolyte concentration in a sample fluid utilizing ion selective electrodes are known. Typically the measurement apparatus includes, an electrometer connecting a pair of chemical half cells including a first ISE sensor half cell and a reference electrode half cell. For determining potassium concentration of a patient serum sample, for example, the sensor half cell includes an electrode and membrane separated by a fill solution of known potassium concentration. The membrane has an affinity for potassium ions and is typically made from an ion sensitive polypeptide composition, e.g. valinomycin in polyvinyl chloride. When the sensor half cell membrane is brought in contact with the fluid sample, the sensor membrane forms very specific complexes with the potassium ions in the sample. A voltage or potential is developed across the membrane that is proportional to the unknown potassium concentration in the sample. Following the Nernst Equation, by knowing the ion concentration on the fill solution side of the sensor membrane and measuring the potential across the sensor membrane, it is possible to calculate the unknown ion concentration on the opposite or sample side of the membrane.
A reference half cell is similar in its construction to the ISE sensor half cell except that the reference membrane is generally formed of a non-ion specific material, typically ceramic or glass frit. The reference half cell is designed to maintain a relatively constant potential and is used to complete the electrical circuit through the unknown sample.
Centrifugal type clinical analyzers are used to perform a variety of clinical tests on a fluid sample, such as whole blood, plasma or serum. In accordance with the above-identified pending application, the analyzer uses centrifugal force to separate whole blood; measure reagents and plasma volumes; and to complete all steps required for spectrophotometric analysis. The system is based on a two dimensional centrifugation process in which a multi-chambered cartridge or cassette containing the sample and liquid reagents is centrifuged at 500 G's in two planes oriented at right angles to one another. The plastic multi-chambered test cartridge itself is of a very small size having dimensions generally similar to an ordinary credit card size and being between 1/4 to 1/2 of an inch in thickness.
A major object of this invention is to provide an ion-selective electrode system including an ISE sensor half cell and a reference half cell which are incorporated into the small sized cassette for use on the centrifugation analyzer equipment. To satisfy this object, considerable miniaturization for the ISE sensor electrode, and the reference electrode, had to be achieved.
Conventional reference electrodes have generally been of two popular types including the Hg/Hg.sub.2 Cl.sub.2 (calomel) type and the Ag/AgCl type. These reference electrodes form a stable electrochemical half cell when placed in contact with a known reference solution of known chloride ion concentration.
A key problem in making a reference electrode is to provide both an electrical connection and a liquid junction between a reference liquid solution in the reference electrode and the sample being tested. The interface between the liquids should be sufficiently permeable to permit appropriate ion migrations on both sides of the junction with a low characteristic junction resistance. The liquid junction must also provide a practical barrier to fluid flow to prevent sample contents from entering into the electrode itself and thereby changing the composition of the reference fill solution and therefore the reference potential. Prior art reference electrodes have used glass frits and fibers to form the liquid junction interface between the reference solution and the sample.
A major problem with the glass frit liquid junction barriers is that contamination of the reference liquid fill solution often occurs due to permeability. Changes or drift in the reference half cell potential may result which require that the half cell be re-calibrated often. Attempts to overcome the potential drift have included a circulating fresh reference liquid solution through the reference half cell on the electrode side of the reference membrane. Expensive and elaborate equipment modifications are required to provide a flow system for the reference fill solution.
In the present design context, size constraints built into the test cartridge for use in the centrifugation equipment determined that a miniaturized reference electrode was needed, having a liquid junction orifice of approximately 0.020 of an inch in diameter.
A major design difficulty was to develop a membrane that could physically separate the sample from the reference fill solution but which would form an electrical connection and liquid junction therebetween characterized by low reference junction resistance to provide improved electrochemical sensing with respect to the solution.
In this design context, glass frit or fiber membranes were poor candidates for filling such a small orifice at the liquid junction of the reference electrode. A number of other micro porous filter membranes were considered to form the reference membrane, such as nylon filters, but it was difficult to determine how these membranes could be glued, tacked or welded into the reference electrode orifice. Moreover, many prior art membranes were unable to restrict flow or permeability by an amount sufficient to minimize the sample contamination of the reference cell fill fluid without disadvantageously increasing the minimum junction resistances of the reference half cell.
It is known from U.S. Pat. No. 3,333,626, for example, that various cellulose acetate membrane forming compositions may be used for making a cast planar membrane having appropriate membrane characteristics for kidney dialysis. More particularly, the '626 patent describes a method for making advantageous screen membranes by: mixing cellulose acetate in a major organic solvent; adding a second diluent solvent; and adding a metal salt to provide a substantially monophasic solution. The solution is cast onto a polished plane surface to form a thin film. Thereafter, the solvents are removed from the composition by evaporation at a temperature less than 50.degree. C. During the evaporation, a microphase separation of the two solvent based systems occurs, providing a cellulose acetate web having discreet metal ions or salt particles positioned or imbedded therein. Upon full evaporation and drying of the film membrane, the metal salts are removed from the film by contacting the film with a solvent for the metal salt thereby removing it from the porous membrane into solution. This leaves a plurality of micropores in the cellulose acetate web. The '626 patent does not describe or suggest a method for securely attaching the membrane made therein to an orifice of the type presented in a reference electrode, much less a tiny orifice of about 0.020 of an inch in diameter, as required for the miniature reference electrodes contemplated herein. The overall miniature reference electrode half cell for use in the centrifugal clinical analyzer and test cartridge was required to be approximately the size of a 1/4 to 1/2 inch screw.
In order to overcome the deficincies in prior art reference electrodes, it is an object of the present invention to provide a miniaturized reference electrode adapted for use in a test cartridge or cassette device used in a centrifugation clinical analyzer.
It is another object of the present invention to provide a new and improved miniaturized reference electrode including a low impedence liquid junction membrane capable of providing an electrical connection and a liquid junction between a reference liquid solution in the electrode and a solution to be tested.
It is a further object of the present invention to provide a new and improved reference electrode liquid junction membrane which does not permit, or substantially limits contamination of the reference liquid solution in use.
It is another object of the present invention to provide a method for making a new and improved reference electrode membrane and for attaching the reference membrane to the reference electrode half cell.